Rap1b is a 21 kDa low molecular weight GTP binding protein initially identified and still best known for its capacity to reverse ras transformed cells. Because of this, it has been largely considered to be a regulator of ras activity. More recent work suggests that rap has signaling properties of its own in some cells and regulators of rap G protein function - guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and putative downstream effectors - have been identified. More importantly, rap appears to undergo a G protein cycle in many cells, with reversible binding of GTP and GDP. In blood platelets, rap is an abundant G protein. Rap is activated when platelets are stimulated by a variety of agonists, including ADP, collagen, and thrombin. Rap is also a substrate for cAMP-dependent protein kinase and is phosphorylated in platelets treated with agents that increase intracellular levels of cAMP. The overall aim of the work in this application is to use a rap1b deficient mouse developed in our laboratory to examine the function of rap in platelets, both during transduction of the ADP, collagen, and thrombin signals as well as during platelet inhibition by cAMP. The hypothesis to be tested is that rap1b is a bidirectional signal regulator in platelets, participating in agonist-induced signaling pathways and in the inhibition of platelets by cAMP. To determine the role of rap1b in mouse platelets, we will 1) compare platelet shape change, secretion, and aggregation in rap1b-/-, rap1b+/-, and rap1b+/+ platelets over a range of concentrations of ADP, epinephrine, thrombin, and other receptor-mediated agonists, 2) identify and characterize rap1b effectors in platelets by comparing serine/threonine phosphoproteins, GTP phosphoproteins, MAP kinase activation, and B-raf activation in rap1b-/- and rap1b+/+ platelets, and 3) examine platelet nhibition by cAMP and by nitric oxide in rap1b-/- and rap1b+/+ platelets. If rap functions as a bidirectional regulator, these studies should demonstrate that rap1b-/- platelets have both a defect in platelet aggregation and in the inhibition of platelet activation by cAMP. The results of these studies should provide new understanding of the role of rap1b in platelets and in cells in general. In addition, we should be able to use the deficient mice to identify signaling pathways involved in mediating the effects of rap1b.